Bone is a dynamic tissue, and homeostasis in the adult skeleton requires a balance between bone resorption and bone formation. Osteoclasts and osteoblasts play a key role in this balance, with osteoclasts initiating bone resorption and osteoblasts synthesizing and depositing new bone matrix. Imbalances in bone homeostasis are associated with such conditions as osteoporosis, Paget""s disease, and hyperparathyroidism.
The activities of osteoclasts and osteoblasts are regulated by complex interactions between systemic hormones and the local production of growth factors and cytokines. Calcitonin, a peptide hormone secreted by the thyroid and thymus of mammals, plays an important role in maintaining bone homeostasis. Calcitonin inhibits bone resorption through binding and activation of a specific calcitonin receptor on osteoclasts (The Calcitonins-Physiology and Pharmacology Azria (ed.), Karger, Basel, Su., 1989), with a resultant decrease in the amount of calcium released by bone into the serum. This inhibition of bone resorption has been exploited, for instance, by using calcitonin as a treatment for osteoporosis, a disease characterized by a decrease in the skeletal mass often resulting in debilitating and painful fractures. Calcitonin is also used in the treatment of Paget""s disease where it provides rapid relief from bone pain, which is frequently the primary symptom associated with this disease. This analgesic effect has also been demonstrated in patients with osteoporosis or metastatic bone disease and has been reported to relieve pain associated with diabetic neuropathy, cancer, migraine and post-hysterectomy. Reduction in bone pain occurs before the reduction of bone resorption.
Salmon calcitonin has been shown to be considerably more effective in arresting bone resorption than human forms of calcitonin. Several hypotheses have been offered to explain this observation: 1) salmon calcitonin is more resistant to degradation; 2) salmon calcitonin has a lower metabolic clearance rate (MCR); and 3) salmon calcitonin may have a slightly different conformation, resulting in a higher affinity for bone receptor sites.
Despite the advantages associated with the use of salmon calcitonin in humans, there are also disadvantages. For treatment of osteoporosis, for instance, the average cost can exceed $75 a week and involve daily prophylactic administration for 5 or more years. In the United States, calcitonin must be administered by injection, and since the disease indications for this drug are not usually life threatening, patient compliance can be low. Resistance to calcitonin therapy may occur with long-term use. What triggers this resistance or xe2x80x9cescape phenomenonxe2x80x9d is unknown (see page 1093, Principles of Bone Biology, Bilezikian et al., (eds.) Academic Press, NY; Raisz et al., Am. J. Med. 43:684-90, 1967; McLeod and Raisz, Endocrine Res. Comm.8:49-59, 1981; Wener et al., Endocrinology. 90:752-9, 1972 and Tashjian et al., Recent Prog. Horm. Res. 34:285-303, 1978). Use of calcitonin mimetics, either in place of native calcitonins or in rotation with native calcitonins, would help avoid resistance to such treatment during long-term use. In addition, some patients develop antibodies to non-human calcitonin, calcitonin mimetics would be useful for such patients.
What is needed in the art are alternative methods of inhibiting bone resorption. The present invention fulfills these and other needs.
The present invention provides isolated compounds that are useful as calcitonin mimetics. As used herein, the term xe2x80x9ccalcitonin mimeticxe2x80x9d refers to a compound with the ability to mimic the effects generated by calcitonin""s interaction with its receptor and its signal transduction pathway and, by such interaction, stimulate G-protein-mediated activation by adenyl cyclase.
Within one aspect the invention provides a compound of formula I: 
wherein R1 and R2 are each members independently selected from the group consisting of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenyls having from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl, substituted alkylaryl, carbocyclic ring, substituted carbocyclic ring, heterocyclic ring, substituted heterocyclic ring, and combinations thereof, the combinations are fused or covalently linked and the substituents are selected from the group consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; R3 is a 2,5 disubstituted aryl; R4 and R5 are each independently selected from the group consisting of hydrogen and alkyls having from 1 to 6 carbon atoms, or taken together from a ring selected from the group consisting of saturated or unsaturated five-member rings, saturated or unsaturated six-member rings and saturated or unsaturated seven-member rings; Z and X are each independently selected from the group NH, O, S, or NR, wherein R is a lower alkyl group of from 1 to 6 carbon atoms; n and m are each independently an integer from 0 to 6. Within one embodiment R1 is selected from the group consisting of phenyl, substituted phenyl, benzyl, substituted benzyl, naphthylmethyl, substituted naphthylmethyl, indolymethyl, and substituted indolymethyl; R2 is selected from the group consisting of alkyls of from 1 to 6 carbon atoms, alkenyls of from 1 to 6 carbon atoms, benzyl, substituted benzyl, naphthylmethyl, and substituted naphthylmethyl; wherein substituents are selected from the group consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; and R4 and R5 are hydrogen; Z is O; and X is NH. Within a related embodiment R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl; and R2 is ethyl, allyl, benzyl or 2-naphthylmethyl. Within another embodiment the compound has the formula: 
wherein, R1 and R2 are each independently selected from the group consisting of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenyls having from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl, substituted alkylaryl, carbocyclic ring, substituted carbocyclic ring, heterocyclic ring, substituted heterocyclic ring, and combinations thereof, the combinations are fused or covalently linked and the substituents are selected from the group consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; and S1, S3 and S4 are each independently selected from the group consisting of hydrogen, halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl. S2 and S5 are each independently alkyl or aryl. Within one embodiment R1 is selected from the group consisting of phenyl, substituted phenyl, benzyl, substituted benzyl, naphthylmethyl, substituted naphthylmethyl, indolymethyl, and substituted indolymethyl; R2 is selected from the group consisting of alkyls having from 1 to 6 carbon atoms, alkenyls having from 1 to 6 carbon atoms, benzyl, substituted benzyl, naphthylmethyl, and substituted naphthylmethyl; wherein the substituents are selected from the group consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl and S2 and S5 are t-butyl. Within a related embodiment R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl; R2 is ethyl, allyl, benzyl or 2-naphthylmethyl; and S2 and S5 are t-butyl.
Within another aspect, the invention provides a pharmaceutical composition comprising an effective amount of a compound as described above in a pharmaceutically acceptable carrier.
Within another aspect the invention provides a method for treating a bone-related disorder, comprising administering to a subject suffering from such disorder an effective amount of calcitonin mimetic compound as described above. Within a related embodiment the bone-related disorder is selected from the group consisting of osteoporosis, Paget""s disease, hyperparathyroidism, osteomalacia, periodontal applications (bone loss), hypercalcemia of malignancy and hypercalcemia of infancy.
Within another aspect the invention provides a method of inhibiting bone resorption comprising administering to a subject in need of such inhibition an effective amount of a calcitonin mimetic compound as described above.
Within yet another aspect the invention provides a method for providing an analgesic effect comprising administering to a subject in need of such an effect an effective amount of a calcitonin mimetic compound as described above. Within a related embodiment the analgesic effect provides relief from bone pain.
Within another aspect the invention provides a method for treating conditions associated with inhibiting gastric secretion comprising administering to a subject in need of such inhibition an effective amount of a calcitonin mimetic compound as described above. Within a related embodiment the conditions associated with inhibiting gastric secretion is a gastrointestinal disorder.
These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawings.
Abbreviations
The following abbreviations are used herein: Boc, t-butoxycarbonyl; DCM, dichloromethane; DME, dimethoxyethane; DMF, dimethylformamide; EtOAc, ethyl acetate; Fmoc, fluorenylmethoxycarbonyl; TFA, trifluoroacetic acid.
All references cited herein are incorporated by reference in their entirety.
The calcitonin mimetics which are useful in the present invention are those compounds with the ability to mimic the interaction of calcitonin with its receptor and, by such mimicry, to stimulate G-protein-mediated activation of adenyl cyclase or activation of CRE by an alternative signal transduction pathway. These mimetics are represented by the general formula: 
In this formula, R1 and R2 are each independently hydrogen, alkyl groups having from 1 to 6 carbon atoms, alkenyl groups having from 1 to 6 carbon atoms, an aryl group, or alkylaryl groups, where the alkyl portion may have 1 to 6 carbon atoms and the aryl portion represents an aryl group, a substituted aryl group, a carbocyclic ring, a substituted carbocyclic ring, a heterocyclic ring, a substituted heterocyclic ring, or combinations thereof. The combinations can be fused or covalently linked. In certain preferred embodiments R1 is substituted or unsubstituted phenyl, benzyl, naphthylmethyl or indolymethyl. R2 is an alkyl or alkenyl having from 1 to 6 carbon atoms, substituted or unsubstituted benzyl or naphthylmethyl. In certain particularly preferred embodiments R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl. R2 is ethyl, allyl, benzyl, or 2-naphthylmethyl.
R3 represents substituted and unsubstituted aryl groups, carbocyclic rings, heterocyclic rings, or combinations thereof. The combinations can be fused or covalently linked. Within certain preferred embodiments R3 is a 2,5 disubstituted aryl. Preferably the substitutions are aryl or alkyl. Within a preferred embodiment R3 is 2,6-di-t-butyl-phenyl.
R4 and R5 are each independently hydrogen, alkyl groups having from 1 to 6 carbon atoms. Within certain embodiments R4 and R5 can be joined together to form a ring which is a four-, five-, six- or seven-member ring, saturated or unsaturated. For those embodiments in which the ring is unsaturated, the ring can be an heteroaromatic ring (e.g., pyrimidyl, imidazyl). Within certain preferred embodiments R4 and R5 are hydrogen.
Z and X each independently represent either NH, NR, O, or S, in which R is a lower alkyl group of from one to six carbon atoms. In preferred embodiments, Z represents O and X represents NH. The symbols n and m each represent independently, integers from zero to six.
As used herein, the term xe2x80x9calkylxe2x80x9d refers to a saturated hydrocarbon radical which may be straight-chain or branched-chain (for example, ethyl, isopropyl, or t-butyl), or cyclic (for example cyclobutyl, cyclopropyl or cyclopentyl). Preferred alkyl groups are those containing 1 to 6 carbon atoms.
The term xe2x80x9calkenylxe2x80x9d refers to an unsaturated hydrocarbon radical which may be a straight-chain, branched-chain or cyclic. Examples of alkenyls include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl, as well as dienes and trienes of straight, branched or cyclic chains and the like. Preferred alkenyl groups are those containing 1 to 6 carbon atoms.
The term xe2x80x9carylxe2x80x9d refers to an aromatic substituent which may be a single ring or multiple rings which are fused together, linked covalently or linked to a common group such as an ethylene or methylene moiety. The aromatic rings may each contain heteroatoms, preferred heteroatoms are N, S or O. Examples of aryl groups include phenyl, benzyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl, thienyl, pyridyl and quinoxalyl. Additionally, the aryl groups may be attached to other parts of the molecule at any position on the aryl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pyridyl, 3-pyridyl and 4-pyridyl).
Heterocyclic rings contain at least one heteroatom selected from N, O and S. Examples of carbocyclic and heterocyclic rings include cyclohexyl, cyclohexenyl, piperazinyl, pyrazinyl, morpholinyl, imidazolyl, triazolyl and thiazolyl.
The terms xe2x80x9csubstituted alkylxe2x80x9d, xe2x80x9csubstituted alkenylxe2x80x9d, xe2x80x9csubstituted alkylarylxe2x80x9d, xe2x80x9csubstituted arylxe2x80x9d, xe2x80x9csubstituted carbocyclic ringxe2x80x9d, xe2x80x9csubstituted heterocyclic ringxe2x80x9d xe2x80x9csubstituted phenylxe2x80x9d, xe2x80x9csubstituted benzylxe2x80x9d, xe2x80x9csubstituted naphthylmethylxe2x80x9d and xe2x80x9csubstituted indolymethylxe2x80x9d refer to the above alkyl, alkenyl, alkylaryl, carbocyclic ring, heterocyclic ring, aryl, phenyl, benzyl, naphthylmethyl and indolymethyl groups substituted by one or more, preferably one, halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl. Examples include 4-ethoxybenzyl, 1-ethyl-indolylmethyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl or 2-naphthylmethyl.
All numerical ranges in this specification and claims are intended to be inclusive of their upper and lower limits.
In one group of preferred embodiments, the calcitonin mimetics are represented by the formula: 
In this formula, the symbols R1 and R2 have the meaning provided above. The symbols S1, S3 and S4 each independently represent a substituent on the attached aromatic ring which is hydrogen, halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl. The symbols S2 and S3 each represent an aryl or alkyl. In certain preferred embodiments R1 is substituted or unsubstituted phenyl, benzyl, naphthylmethyl or indolymethyl. R2 is an alkyl or alkenyl having from 1 to 6 carbon atoms, substituted or unsubstituted benzyl or naphthylmethyl. In certain particularly preferred embodiments, S5 and S2 are t-butyl, R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl and R2 is ethyl, allyl, benzyl, or 2-naphthylmethyl.
The calcitonin mimetics used in the present invention can be prepared using commercially available materials. A general synthetic scheme for preparing molecules of Formula I wherein R4 and R5 are hydrogen, using methodologies known in the art, is provided herein. 
In a typical preparation, 100 mg of p-methylbenzhydrylamine (MBHA) resin (0.81 meq/g, 100-200 mesh) was contained within a sealed polypropylene mesh packet. Following neutralization with 5% diisopropylethylamine (DIEA) in dichloromethane (DCM), the resin was washed with DCM. The first protected amino acid was coupled using hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DICI) in DMF. Following removal of the amino protecting group, the mesh packet was shaken overnight in a solution of 0.1 M trityl chloride in DCM/DMF (9:1) in the presence of DIEA. Completeness of the trityl coupling was verified using the bromophenol blue color test as described in Krchnak et al., (Coll. Czech. Chem. Commun. 53:2542, 1988, and repeated as necessary.
N-alkylation was then performed by treatment of the resin packet with 1 M lithium t-butoxide in THF (20xc3x97) for 15 min, as described by D{grave over (rner)}, et al., (Bioorg. Med. Chem. 4:709, 1996). Excess base was then removed by decantation, followed by addition of the individual alkylating agent in DMSO (20xc3x97, 0.1M). The solution was vigorously shaken for 2 h at room temperature. This step is normally repeated three times for methyl iodide, and five times for the other alkylating agents. Small aliquots of the resin can be cleaved to determine the completeness of this step. The trityl group was removed with 2% TFA in DCM (2xc3x9710 min).
The isocyanate of the incoming primary amine (or aniline) was performed by slowly adding a solution of the primary amine (0.3M in DCM, 24xc3x97 over the resin substitution) and DIEA (48xc3x97) dropwise to solution of 0.1M triphosgene (8xc3x97) in DCM. It is known in the art that the reaction does not proceed through the isocyanate for secondary amines. The packet was washed, neutralized and the isocyanate solution added and shaken for 1 hour at RT. Following decantation, the isocyanate solution was quenched with 10% NH3 in DMF. The resin was washed with DCM, 0.05% NH3 in DMF, MeOH, DCM, and MeOH.
The product was cleaved from the resin with anhydrous HF by the procedures of Houghten et al., (Int. J. Pep. Prot. Res. 27:673, 1986), in the presence of anisole. The product was extracted with 50% ACN/H2O and lyophilized, followed by relyophilization from 50% acetonitrile.
The compounds of the invention can be administered to warm blooded animals, including humans, to mimic the interaction of calcitonin with its receptor in vivo. Within one aspect, calcitonin mimetics of the present invention are contemplated to be advantageous for use in therapeutic defects for which calcitonin is useful. In particular, the calcitonin mimetics are useful for the regulation of bone metabolism and reduction of serum calcium. The calcitonin mimetics of the invention can be administered to warm blooded animals, including humans, to mimic the interaction of calcitonin with its receptor in vivo. Thus, the present invention encompasses methods for therapeutic treatment of bone-related disorders. Such bone-related disorders include, but are not limited to, osteoporosis, Paget""s Disease, hyperparathyroidism, osteomalacia, periodontal defects (bone loss), hypercalcemia of malignancy, idiopathic hypercalcemia of infancy, and other related conditions. Calcitonin mimetics are also contemplated to be advantageous as analgesics, in particular for relief of bone pain. Calcitonin mimetics are further contemplated to be advantageous in inhibiting bone resorption. The calcitonin mimetics of the present invention can also be used to inhibit gastric secretion in the treatment of acute pancreatitis and gastrointestinal disorders. The methods of the present invention may be used to treat these conditions in their acute or chronic stages.
Pharmaceutically or therapeutically effective amounts of calcitonin mimetics of the present invention can be formulated with pharmaceutically or therapeutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal administration or the like, according to conventional methods. Formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and maybe provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example. Slow or extended-release delivery systems, including any of a number of biopolymers (biological-based systems), systems employing liposomes, and polymeric delivery systems, can also be utilized with the compositions described herein to provide a continuous or long-term source of the calcitonin mimetic. Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use. The term xe2x80x9cpharmaceutically or therapeutically acceptable carrierxe2x80x9d refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient. One skilled in the art may formulate the compounds of the present invention in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Preferably such compounds would be administered orally or parenterally.
As used herein, a xe2x80x9cpharmaceutically or therapeutically effective amountxe2x80x9d of such a calcitonin mimetic is an amount sufficient to induce a desired biological result. The result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an effective amount of a calcitonin mimetic is that which provides either subject relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer. In particular, such an effective amount of a calcitonin mimetic results in reduction in serum calcium, inhibition of bone resorption, inhibition of gastric secretion or other beneficial effect. Effective amounts of the calcitonin mimetics can vary widely depending on the disease or symptom to be treated. The amount of the mimetic to be administered, and its concentration in the formulations, depends upon the vehicle selected, route of administration, the potency of the particular mimetic, the clinical condition of the patient, the side effects and the stability of the compound in the formulation. Thus, the clinician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients. Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the patient, and other factors evident to those skilled in the art. Estimation of appropriate dosages effective for the individual patient is well within the skill of the ordinary prescribing physician or other appropriate health care practitioner. As a guide, the clinician can use conventionally available advice from a source such as the Physician""s Desk Reference, 48th Edition, Medical Economics Data Production Co., Montvale, N.J. 07645-1742 (1994). Typically a dose will be in the range of 0.1-100 mg/kg of subject. Preferably 0.5-50 mg/kg. Doses for specific compounds may be determined from in vitro or ex vivo studies on experimental animals. Concentrations of compounds found to be effective in vitro or ex vivo provide guidance for animal studies, wherein doses are calculated to provide similar concentrations at the site of action.
Well established animal models are available to test in vivo efficacy of calcitonin mimetics. For example, the hypocalcemic rat model can be used to determine the effect of synthetic calcitonin mimetics on serum calcium, and the ovariectomized rat or mouse can be used as a model system for osteoporosis. Bone changes seen in these models and in humans during the early stages of estrogen deficiency are qualitatively similar. Calcitonin has been shown to be an effective agent for the prevention of bone loss in ovariectomized humans and also in rats (Mazzuoli, et al., Calcif. Tissue Int. 47:209-14, 1990; Wronski, et al., Endocrinology 129:2246-50, 1991).
Only those compounds which retain calcitonin-like activity, as assayed by a CRE-luciferase assay, for example, are within the scope of this invention. The calcitonin receptor is a member of the G-protein receptor family and transduces signal via activation of adenylate cyclase, leading to elevation of cellular cAMP levels (Lin, et al., Science 254:1022-4, 1991). This assay system exploits the receptor""s ability to detect other molecules, not calcitonin, that are able to stimulate the calcitonin receptor and initiate signal transduction.
Receptor activation can be detected by: (1) measurement of adenylate cyclase activity (Salomon, et al., Anal. Biochem. 58:541-8, 1974; Alvarez and Daniels, Anal. Biochem. 187:98-103, 1990); (2) measurement of change in intracellular cAMP levels using conventional radioimmunoassay methods (Steiner, et al., J. Biol. Chem. 247:1106-13, 1972; Harper and Brooker, J. Cyc. Nucl. Res. 1:207-18, 1975); or (3) use of a CAMP scintillation proximity assay (SPA) method (Amersham Corp., Arlington Heights, Ill.). While these methods provide sensitivity and accuracy, they involve considerable sample processing prior to assay, are time consuming, may involve the use of radioisotopes, and would be cumbersome for large scale screening assays.
An alternative assay system (described in WO96/31536) involves selection of substances that are able to induce expression of a cyclic AMP response element (CRE)-luciferase reporter gene, as a consequence of elevated cAMP levels or other signaling pathways, such as stimulation of Ca++/Ip3 pathway leading to CRE induction, in cells expressing a calcitonin receptor, but not in cells lacking calcitonin receptor expression. Such cells could include, for example, Boris/KS10-3 (expressing hamster calcitonin receptor and a CRE-luciferase reporter gene in baby hamster kidney cells (BHK 570 cells)) or Hollex 1 or Hollex 2 (expressing human calcitonin receptor and a CRE-luciferase reporter gene in BHK cells, as described in WO96/31536) or KZ10-20-48/pLJ6-4-25, which expresses the human glucagon receptor and a CRE-luciferase reporter gene in BHK cells. The human glucagon receptor is another member of the G-protein-coupled receptor that transduces signal through adenylate cyclase-mediated elevation of cAMP. PTH can be used as a control as well.
This CRE-luciferase assay measures the end result of a multi-step signal transduction pathway triggered when a calcitonin mimetic stimulates the G-coupled calcitonin receptor. The complexity of this pathway provides multiple mechanisms for induction of luciferase transcription at points that are downstream of the calcitonin receptor, and therefore may not be calcitonin receptor-specific (e.g., forskolin""s direct activation of adenylate cyclase). Any response triggered by non-specific inducers is eliminated by counter screening using the calcitonin receptor-negative cell lines described above.